Results: The Colony PCR of the Cytosine Deaminase (CD) did not work out. The detectable bands at around 700 bp correspond to CFP. Therefore, BioBrick production of the CD must be repeated. The following three bands represent the controls. Three different controls were used. The first corresponds to pSB1C3_CFP, the second to BAP_587 and third control is the negative control. Unfortunately now bands can be detected in the postive controls. A faint band can be seen in the negative control which is the only lane in which no band should be detectable.
The following bands after the three controls correspond to the approaches of the Z34C Viralbrick production. Some bands in the lower and the upper lanes show positive results. The corresponding inoculated overnight culture were centrifuged and prepared in order to perform a Mini-Prep tomorrow.

Comment: hmm, seems that the CD-assambly didn't work at all.. Should i try it again or are we gonna skip it for good due to other priorities?(kira)
No, we cannot skip it. Can we change something else?? Any ideas what we can alter in the protocol? Because problem is that we are still waiting for the tk... so we should aswell focus on the other prodrug activating enzmye!! (Bea)

Testtransduction of the final modified capsid coding constructs

Investigator: Adrian

Aim of the experiment:
During the project 22 silent nucleotide exchange mutations were introduced into the capsid coding construct to make it compatible with the RFC standards and to have single cutting restriction enzymes flanking the 453 and the 587 loop sequence. Two point mutations had to be dismissed, because either a first test transduction showed that the construct was not working anymore or the insertion of the synthesized gene posed serious problems, because the restriction enzyme did not work.Finally, we have a construct that is shown to produce infectious particles comparable to the current AAV systems and carries 20 point mutations. Now we can announce that the Adeno-associated Virus is compatible to the RFC standard and the idea to replace the loop sequences via ViralBricks works!

Impression from the moment:

The construct with the insertion of the Rep and the Cap synthesis worked! Reintroduction of the KpnI restriction site recovered the functionality of the viral genome!

Miniprep of serveral constructs

Investigator: Stefan

Glycerol stocks were prepared:

B430 = pCerulean_ZEGFR:1907_Longlinker_VP2/3

B431 = pCerulean_CFP_Middlelinker_VP2/3_insCap

B432 = pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap

Mini-Prep was performed according to the standard protocol

P527 = pCerulean_ZEGFR:1907_Longlinker_VP2/3 c = 230,61 ng/µl

P528 = pCerulean_CFP_Middlelinker_VP2/3_insCap c = 290,81 ng/µl

P529 = pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap c = 269,39 ng/µl

Several other constructs were preped, but need to be confirmed before being added to plasmid/glycerol stock.

Gel-Extraction:
Gel-extraction was performed according to standard protocol.
c(P320) = 0,8 ng/µl

Ligation:

1 µl T4 DNA ligase

1 µl 10x Buffer

8 µl DNA mix (vector + insert)

Amounts of DNA used:

c(P320) = 7,73 µl

c(P455) = 0,27 µl

c(P320) = 7,19 µl

c(P463) = 0,81 µl

Incubation time: 45 minutes at room temperature.

Transformation:
Transformation was performed according to standard protocol using XL1b cells and Chloramphenicol as antibiotic.

2x repetition of CD biobrick

Investigator: Kira

Ingredients

CD sample

5X Phusion HF buffer

10 µl

10 mM dNTP mix

1µl

forward primer: O158

2,5µl

reverse primer: O159

2,5 µl

DNA Template (1:100 dil)

0,5 µl

DMSO

0 µl

Phusion Polymerase

0,5 µl

H2O

33µl

Total volume

50 µl

PCR program:

Cycles

Temperature

Time

98°C

1

10x

98°C

15"

58°C

25"

72°C

40"

17x

98°C

15"

65°C

25"

72°C

40"

1x

72°C

5'

Hold 4°C

Digestion of plasmid backbone:

c (pSB1C3) = 151, 1 ng/ µl

Components

vector Volume/µL

DNA 1 µg

6,0 µl

BSA (100x)

0,2 µl

Buffer no. 4 (10x)

2,0 µl

Enzyme 1 XbaI

0,5 µl

Enzyme 2 AgeI HF

0,5 µl

H2O

10,8 µl

Total volume

20

incubation @ 37 C for approx. 6 h

1% agarose gel

According to the gel results, PCR was repeated twice.. But both tries revealed the same unexpected results. Insert from the last cloning procedure was found and used for the ligation and further transformation.

Transformation was performed according to the standard protocol and the cells were spread on the agar plate containing Chloramphenicol.

126. labday 21.09.2010

Trafo evalutation of CD biobrick plate

Investigator: Kira

the agar plate was checked at approx. 4 pm and there were no colonies visible. Taking in account that the plate was incubated from 10.30 pm till 8 am @ 37C due to the electricity failure, the plate was put back @ 37C at 4.30 pm

Comment: the plate was picked this morning by a lab member and put in the coldroom

Continuation of cloning of pCerulean_Affibody_VP2/3 and pSB1C3_Affibody_middlelinker_GFP_his

Investigator: Jessica

Comment:Affibody_VP2/3 will be cloned in pCerulean and Affibody_middlelinker will be cloned together with GFP_His in pSB1C3

P407= pCerulean_CFP_middlelinker c=482,76 ng/µl

P516= pSB1C3_ZEGFR:1907_VP2/3 clone 1 c= 191,6 ng/µl

P290= pSB1C3_Affibody_Middlelinker clone 1 c= 227,4 ng/µl

P518= pSB1C3_GFP_RFC25_upper band clone 1 c= 138,3 ng/µl

P520= pSB1C3_GFP_RFC25_lower band clone 1 c= 141,6 ng/µl

Digestion:

components

P516

P518

P520

DNA

10

10

10

BSA (10x)

2

2

2

Buffer 4 (10x)

2

2

2

EcoRI

0,8

-

-

AgeI

0,8

-

-

MscI

0,8

-

-

NgoMIV

-

0,8

0,8

SpeI

-

0,8

0,8

H2O

3,6

4,4

4,4

Total volume

20

20

20

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 120 Volt

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

P516= c= ng/µl

P518= c= ng/µl

P520= c= ng/µl

T4 Ligation:

The Ligation was performed as following:

Vector Volume: µl

Insert Volume: µl

1µl T4-Ligase buffer (10x)

8µl (Vector + Insert) mix

1µl T4-Ligase

Incubating for 40 minutes.

Transformation:

Trafo was performed according to the standard protocol (XL1blue). The cells were plated on a agar plate with Kana/Cm

Sequencing result of pSB1C3_EGFP_His upper and (!) lower band

Investigator: Jessica 1. pSB1C3_EGFP_His suffix:

2. pSB1C3_EGFP_His prefix:

results look in both plasmids and are used for cloning of pSB1C3_Affibody_middlelinker_GFP_His

New aliquots (60µl) of XL1blue can be used ! (Jessica)

127. labday 22.09.2010

Sequencing confirmed a correct sequence for clone 11.4 (453 Z34C). Clone 12.1 (587 Z34C)has several missing bases at the 3' end of the insert, clone 13.2 has an a -> c mutation and a c insertion in the insert (although the a->c might be a sequencing error.). Clone 14.2 contained a c deletion.

We have several new approaches:

-Correction of the mutations via PCR: We'll use the hybridisation oligo for the side of the insert containing the mutations, the rfc 25 primer on the other side. The amplified insert will be ligated into dephosphorized vector.

-As a backup, SDM-primers will be ordered and should be available next week.

-Also, a new colony pcr with several clones from the last ligation plates will be carried out.

Investigator Patrick
After an over night digestion with BamHI and PvuII at 37°C ....

My dear friend - what do you mean with "..."?? overnight digest at which temperature?? Did you look it up whether the enzymes show star activity at longer digestions or not?? ;-)

The Gelextraction with P514 was performed according to the standard protocol yielding 16,9 ng/ml.
The P542 insert should be 48 bp long and therefore was extracted with a QIAGEN kit able to extract even very small DNA fragments (40 bp) (QiaEX II Gel Extraction Kit (150)).

Expected size of the fragments:
P542: 2112 & 48 bp
P514: 3956 & 48 bp

The 48 bp band of P514 can't be seen clearly on the picture due to the bad resolution.

Comment: After several problems were managed (cloning difficulties, colony PCR troubles, interchanges of samples,...), sequences of the final constructs needed to be verified in order to test the constructs in cell culture.

Sample gallery

pCerulean_CFP_MiddleLinker_ VP2/3_InsCap (P528) forward

pCerulean_CFP_MiddleLinker_ VP2/3_InsCap (P528) reverse

pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_InsCap (P527) forward

pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_InsCap (P527) reverse

pCerulean_6xHis_MiddleLinker_ VP2/3 (P533) forward

pCerulean_6xHis_MiddleLinker_ VP2/3 (P533) reverse

pCerulean_6xHis_MiddleLinker_ VP2/3_insCap (P534) forward

pCerulean_6xHis_MiddleLinker_ VP2/3_insCap (P534) reverse

pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_insCap (P535) forward

pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_insCap (P535) reverse

pCerulean_ZEGFR:1907_SEG_ VP2/3_insCap (P502) reverse

pCerulean_ZEGFR:1907_LongLinker_ VP2/3_insCap (P503) reverse

pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3 (P504) reverse

Comment: Sequences looked well. The 6 samples with VP2/3_insCap will be tested today in cell culture.

Gel apparatus (chamber, tray and comb) for RNA purity gel has to be prepared a day ahead with : water, SDS and NaOH

cDNA synthesis

Investigator: Kira

The yielded DNA was used for cDNA synthesis.
QuantiTect Reverse Transcription (Qiagen) reaction was performed according to the manufacturer protocol.
DNA elimination reaction mix:

components

a431/µl

293/µl

7xgDNA Wipeout Buffer

2

2

Template RNA

1,6

2

RNase-free H2O

10,4

10

Total volume

14

14

Reverse-transcription reaction mix:

components

1xreaction

MM

Quantiscript Reverse Transcriptase

1

3

5x Quantiscript RT buffer

4

12

RT primer Mix

1

3

DNA elimination reaction mix

14

-

Total volume

20

-

incubation for 15 min @ 42C
incubation for 3 min @ 95C

128. labday 23.09.2010

PCR of VB constructs to remove mutations

Investigator Achim

Colony PCR of new clones from the last ligations

Investigator Achim

Sequencing of the previously picked clones revealed that the inserts were successfully cloned into pSB1C3. Sadly, apart from 453 Z34C every other insert contained various mutations (deletions, insertions, base exchanges in different positions). The reasons are unclear, probably difficulties with the fill in reaction or incorrectly synthesized oligos. We picked more clones from the last ligations' plates and carried out a colony pcr on them:

Investigator Patrick
3 Clones were picked in the morning and in each case inoculated 10 ml DYT. Clone 3 didnt grew fast enough. There were not enough bacteria in the eppi even after 8 ml were centrifuged. The mini-prep of clone 1 and 2 was performed according to the standrd protocol yielding the following concentrations:
Clone 1: 36,64 ng/µl
Clone 2: 7,8 ng/µl
Therefore only clone 1 was sent for sequencing (labelling: pat1, primer:VR2) and only this clone received a plasmid number: P569
No test-digestion was performed.
Because all medium was used to receive a sufficient amount of bacteria from clone 1 there was no cell suspension left to prepare a glycerol stock. Therefore again each clone was used to inoculated 10 ml DYT, following a mini-prep tomorrow.

Sequencing results:

Obviously there is something wrong with the backbone or the sequencing.

Comment:The primer O120 was firstly given to the samples. It's a different primer for the Vp123_suffix, which binds to the regulatory-sequence. Because there was no knowledge, if the Vp_ex constucts have this sequence or not, O92 was also given to the samples.

Comment:Because gel-picture showed only DNA within the gel-bags, PCR was repeated.

Comment:Strangely, PCR-products did not run into the gel, altough the PCR was repeated, after first PCR had two reverse primers in the sample. This could be because of a complex with the polymerase.However, ligation will be continued with the DNA within the bags.

hTERT PCR Reaction

Investigator: Kira

@Kira: Do you think that we can (suggestion?) use the A431 for detection of gene expression driven by phTERT promoter?? We should keep in mind that the transduction efficieny will not be that well ;-) (Bea)

Ingredients

Volume / µl

10x PCR buffer

2

10 mM dNTP mix

0,4

50mM MgCl2

0,6

Primer 1

0,5

Primer 2

0,5

cDNA template

1

Taq Polymerase

0,1

H2O

14,9

Total volume

20

PCR program:

Cycles

Temperature / °C

Time

1x

94

3min

30x

94

45sec

51

30

72

1min 30sec

1x

72

10

Hold

4

1% agarose gel:

Evaluation: The agarose gel reveals that all samples show telomerase activity but in different levels.

Repetition: Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Investigator: StefanDigestion of the constructs:

P432 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl

P432 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl

P180 = pSB1C3_pTAV2 (P5)clone 1 c=108,8 ng/µl

P184 = pSB1C3_pAAV_RC (P5TATAless)clone 3 c=176,9 ng/µl

My dear friend, did you mix up the Rep-Cap constructs in the plasmid list? P432 = pAAV_RC_insRepCap_KpnI-back ;)

Components

V P432up /µL

VP180up /µL

V P432down /µL

V P184down /µL

DNA

4,5

14

4,5

11

BSA (10x)

2

2

2

2

Buffer no. 4 (10x)

2

2

2

2

Enzyme 1

XbaI 1

SpeI 1

SpeI 1

XbaI 1

Enzyme 2

EcoRI 1

EcoRI 1

PstI 1

PstI 1

H2O

9,5

9,5

-

3

Total volume

20

20

20

20

Results:Gel-Extraction:
Gel-Ex was performed according to protocol. The following concentrations were measured:

432up c= 25,73 ng/µl

180up c= 9,57 ng/µl

432down c= 27,34 ng/µl

184down c= 17,80 ng/µl

Ligation:
The following DNA mixes were used for ligation:

432up V= 6,3 µl

180up V= 1,7 µl

432down V= 6,93 µl

184down V= 1,07 µl

Transformation:
The transformation was performed according to standard protocol using BL21 cells. They were plated on agar plates containing chloramphenicol and stored in 37°C room overnight.

129. labday 24.09.2010

Sequencing results of VB 13.4

Investigator: Achim

We sent in another clone of the 587 KO Z34C motif to see if it carries the same mutations as the first one. If that had been the case, we could account the mutations to faulty oligos. However, sequencing showed that the second 587 KO Z34C clone is mutated as well, but in different positions. So we either recieved oligos containing various mutations, or the fill-in reaction was faulty in spite of the proofreading activity of the klenow fragment.

Because 587 KO Z34C is one of our more promising approaches, we sent another two clones from our second colony pcr/clone picking round for sequencing: 13.1 and 13.4

Investigator Patrick
Because of the sequencing results of clone 1 a mini-prep was performed only with clone 2 and 3 yielding the following concentrations:
pSB1C3_VP2/3_Capins_587KO-empty clone 2: 195 ng/µl
pSB1C3_VP2/3_Capins_587KO-empty clone 3: 224,74 ng/µl

Sequencing evaluation of VP2/3_Gsat-linker and VP2/3_cap_Gsat-linker

Comment: pSB1C3_VP2/3_cap_Gsat-linker as well as pSB1C3_VP2/3_Gsat-linker can be used for further experiments.

New CD primer ordered

Investigator Stefan

Comment: CD reverse primer was incorrect. A new primer was ordered.

Sequencing of pCerulean_Zegfr:1907_VP2/3

Investigator Hanna

Because I found out, that this plasmid was sent to GATC with the wrong sequencing primers (therefore no sequencing results were available), I sent it again with the GATC_std_CMV-F primer.
--> backbone = pCerulean and not pSB1C3!!! ;)

Repetition: PCR for biobrick production: Cloning RepCap into pSB1C3

Investigator: Stefan

Comment: Construct was cloned mixed RFC10 and RFC25. Therefore, a new approach will be performed consisting of RFC10 only.

Plasmid used:

pAAV_RC_RepCapIns_SDMKpnI (P432)
A 1:1000 dilutionof the plasmid was prepared.

130. labday 25.09.2010

The two clones that were sequenced both contained a deletion in the same position in the insert. We really seem to have bad luck picking clones. Hopefully our PCR attempt works better.

Miniprep of serveral constructs

Investigator: Jessica

Glycerol stocks were prepared:

B489 = pBAD_EGFP_His clone1

B490 = pBAD_EGFP_His clone2

B491 = pBAD_EGFP_His clone3

B492 = pSB1C3_CFP_email clone 1

B493 = pSB1C3_CFP_email clone 2

B494 = pSB1C3_CFP_email clone 3

Mini-Prep was performed according to the standard protocol

P588= pBAD_EGFP_His clone1 c=71,0 ng/µl

P589 = pBAD_EGFP_His clone2 c=74,8 ng/µl

P590 = pBAD_EGFP_His clone3 c=58,3 ng/µl

P591 = pSB1C3_CFP_email clone 1 c=132,7 ng/µl

P592 = pSB1C3_CFP_email clone 2 c=168,9 ng/µl

P593 = pSB1C3_CFP_email clone 3 c=153,6 ng/µl

Test digestion:

Components

Volume P588-590 /µl

Volume P591-593 /µl

Volume P583-585 /µl

DNA

4,5

2,5

2

BSA

1

1

1

Buffer 4 (10x)

1

1

1

XbaI

0,4

0,4

0,4

PstI

0,4

0,4

0,4

H2O

2,7

4,7

5,2

Total volume

10

10

10

Expected size : bp

Comment: test digestion of P591-593 and P583-585 (pSB1C3_P40) is just an attempt because I can't find any sequences in geneious.P583 looks well. P591, P588 and P583 will be sent for sequencing on monday.

Sequencing results of pSb1C3_Affibody_middlelinker_EGFP_His

Comment: there was anywhere a problem in the cloning process. approach will be repeated today. there is pBAD_EGFP_His, affibody_middlelinker will be cloned in, also in pSB1C3_EGFP_His

Sequencing result of pSB1C3_CMV_VP123_capins_clone1

Investigators: Volker, Anna

For test digestion go to labday 24.09.10

Comment: The following primers were used for sequencing: VF2, 4000 for, 4200 rev, 2800 for and 2800 rev. There is a point mutation in the non-coding sequence, which should not influence the performence of the construct, so it can be used for further cloning steps.

Sequencing results: pSB1C3_001_VP2/3_HSPG-KO

Investigator: Hanna

Comment: In order to knock down the natural tropism of the virus, the HSPG knock-out motif (two aa exchanges) was cloned into VP2/3. This fragment can then be used for the VP2 N-terminal fusion or VP1 insertion approaches.

Cloning of pBAD_Affibody_middlelinker_EGFP_His and pSB1C3_Affibody_middlelinker_EGFP_His

Investigator: JessicaComment: There was a mistake in cloning pSB1C3_Affibody_middlelinker_EGFP_His so there is just pSB1C3_EGFP_His. in this plasmid will be cloned Affibody_middlelinker, also in pBAD_EGFP_His

131. labday 26.09.2010

Continuation of cloning of pBAD_Affibody_middlelinker_EGFP_His and pSB1C3_Affibody_middlelinker_EGFP_His

Transformation:

BL21 cells were used - in 37°C at 8.30am

pBAD_Affibody_middlelinker_EGFP_His

pSB1C3_Affibody_middlelinker_EGFP_His

Comment:pSB1C3_Affibody_middlelinker_EGFP_His was not successful (no colonies on plate), will be repeated on monday

Picking clones of different constructs

Investigator: Anna

Clones were picked of:

pBad_Affibody_middlelinker_EGFP_His

pSB1C3_001_RC_InsRepCap_KpnIback_RFC10

Darpin (ReTrafo)

pSB1C3_453_BAP (p402, ReTrafo)

pSB1C3_587_KO_RGD (p390, ReTrafo)

pSB1C3_587_KO_His (p391, ReTrafo)

In addition, preparations for Mini-Preps of several ViralBricks were done:

B289: 587_BAP

B270: 587_KO_BAP

B271: 587_RGD

B272: 453_His

B273: 587_His

B274: 587_KO_empty

B488: 453_Z34C

Comment: Inoculating of 10 mL DYT medium containing 10µL chloramphenicol (except pBad with Amp).
There were no clones on the plate with pSB1C3_Affibody_middlelinker_EGFP_His.

Continuation of cloning of 453 and 587 inserts into pSB1C3_CMV_VP123_capins

Investigator: Achim, Anna

Transformation:

Trafo was performed according to the standard protocol using XL1b cells. The cells were plated on agar plates containing chlorampenicol and stored in the 37°C room.

Colony PCR of remaining 587 constructs

Investigator: Achim, Anna

Comment: New clones of the remaining 587 constructs were picked (from new cloning approach with T4 Ligation and dephosphorylated vector, see labdays 15.09. and 22.09.).

Constructs:

12: 587_Z34C

13: 587_KO_Z34C

14: 587_Z34C_KO_Spacer

Components

Volume /µl

10x Standard Taq Buffer (with MgCl2)

1

MgCl2 (50mM)

0,1

dNTPs

0,2

Primer 1: RFC25 for

0,2

Primer 2: RFC25 rev

0,2

Taq Polymerase

0,05

H2O

8,25

Total volume

10

Cycles

Temperature

Time

95°C

6 min

30x

95°C

25 sec

(Tm -3°C)

60°C

30 sec

1 min/kb

68°C

55 sec

1x

68°C

5 min

Hold 4°C

Clone 13.3, 14.1 and 14.3 will be sent for sequencing.

Sequencing results: pCerulean_Zegfr:1907_VP2/3

Investigator: Hanna

Sequencing results revealed that VP2/3 (unmodified!) was successfully fused to the Affibody (without linker).
Next step: Fusion of VP2/3-HSPG-KO to the C-terminus of the Affibody.

N-terminal VP2-Fusion & VP1 Insertion: Fusion of VP2/3_HSPG-KO

Investigator: Hanna

Comment: In order to target tumor cells with our AAV2 two VP2 fusion strategies were figuered out: The so called VP2-fusion and VP1 insertions. These approaches were successfully cloned and are now tested in cell culture. In order to prevent targeting of other (healthy) cells, the natural tropism of the virus needs to be knocked down. For this purpose a ViralBrick, "587-KO_empty", was designed and cloned into pSB1C3_001_VP2/3_Capins.
Today this VP2/3_HSPG-KO sequence will be fused to the N-terminus of the Affibody, CFP/mVenus and His-Tag of both, the N-terminal fusion to VP2 and the VP1 insertion, approach.

Picking clones of VP1 insertion and VP2 fusion HSPG-KO approaches

MiniPreps of different constructs

The following constructs were prepped according to the standard protocol:

pSB1C3_453_BAP (p402, ReTrafo)

pSB1C3_587_KO_RGD (p390, ReTrafo)

pSB1C3_587_KO_His (p391, ReTrafo)

B289: 587_BAP

B290: 453_RGD

B270: 587_KO_BAP

B271: 587_RGD

B272: 453_His

B273: 587_His

B274: 587_KO_empty

B488: 453_Z34C

B473:

Miniprep of serveral constructs

Investigator: Stefan

Glycerol stocks were prepared:

B489 = pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 1

B490 = pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 2

B491 = pBAD_Affibody_middlelinker_EGFP_His clone 1

B492 = pBAD_Affibody_middlelinker_EGFP_His clone 2

B493 = pBAD_Affibody_middlelinker_EGFP_His clone 3

Mini-Prep was performed according to the standard protocol

P594= pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 1 c= 140,9 ng/µl

P595 = pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 2 c= 117,5 ng/µl

P596 = pBAD_Affibody_middlelinker_EGFP_His clone 1 c= 101,4 ng/µl

P597 = pBAD_Affibody_middlelinker_EGFP_His clone 2 c= 92,9 ng/µl

P598 = pBAD_Affibody_middlelinker_EGFP_His clone 3 c= 92,1 ng/µl

Test digestion:

Components

Volume P594+P595 /µl

Volume P596-598 /µl

DNA

2,5

2,5|

BSA

1

1

Buffer 4 (10x)

1

1

XbaI

0,4

0,4

PstI

0,4

0,4

H2O

4,7

4,7

Total volume

10

10

Comment:

Re-Trafo of pSB1C3_pTAV (P5)

Investigator: Stefan

Comment:After inocculating from glycerol-stock, the plasmid showed strange results during digestion. Therefore a re-trafo will have to be performed to replace the stock.

Repetition: Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Investigator: StefanDigestion of the constructs:

P594 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl

P594 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl

P586 = pSB1C3_pTAV2 (P5)clone 1 c=137,37 ng/µl

P587 = pSB1C3_pAAV_RC (P5TATAless)clone 3 c=191,61 ng/µl

Components

V P594up /µL

VP586up /µL

V P594down /µL

V P587down /µL

DNA

10

14

10

13

BSA (10x)

2

2

2

2

Buffer no. 4 (10x)

2

2

2

2

Enzyme 1

XbaI 1

SpeI 1

SpeI 1

XbaI 1

Enzyme 2

EcoRI 1

EcoRI 1

PstI 1

PstI 1

H2O

9,5

9,5

-

3

Total volume

20

20

20

20

Results:

Comment:P5 and P5TATAless do not fit the expected results. For P5TATAless, a new digestion using the P184 plasmid was performed. For P5 no old plasmid stock was available, therefore a re-trafo will have to be performed.

Picking clones of VP1 insertion and VP2 fusion HSPG-KO approaches

133. labday 28.09.2010

Cloning of the ViralBricks BAP-HSPG-ko and His-HSPG-ko into pSB1C3_VP2/3

Investigator: Bea

Comment: After discussing the finals constructs, we decided to clone HSPG-KO with BAP and His motifs into the N-terminal approaches which means that the plasmids pCerulean_Targeting_VP2/3 contains His and BAP ViralBricks integrated in the 587 loop, respectively. Therefore I started the first cloning step in order to obtain the fully assembled targeting construct which can be cotransfected with the plasmids containing rep and cap genes (with or wihtout HSPG-ko)

Digestion of the constructs:

P604 = pSB1C3_001_587-ko_BAP

P606 = pSB1C3_001_587-ko_His

P525 = pSB1C3_001_VP2/3capins

Components

vP606 /µL

vP525 upstream/µL

vP604 /µL

DNA

21

6

18

BSA (10x)

4

2

4

Buffer no. 4 (10x)

4

2

4

Enzyme 1

BamHI

BamHI

BamHI

Enzyme 2

SpeI

PvuII

PvuII

H2O

9

8

12

Total volume

40

20

40

Loading plan of a 1% agarose gel:

M P525

Loading plan of a 2% agarose gel:

M P604 P606

Expected sizes of the digested products are:

P525= 48,3800 bp

P604= 2216, 81bp

P606 = 2216, 108bp

Results:

After gel extraction has been performed, the 2µL of each ligated plasmids were transformed into BL-21 cells.

Test digestion: ViralBricks in pSB1C3_CMV_VP123_capins

Investigator: Achim

We digested preps of all our 453 Inserts with BamHI & SspI and all our 587 Inserts with SalI and PvuII. That way we get bands of ~50 bp difference between our loop inserts and the wt loop region. (Eg. 453 BAP: 455 with insert, 396 without insert.)

Nomenclature as in previous VB assays.

Clone 11.2 is missing , I forgot to inoculate it.

P580 (pSB1C3_CMV_VP123_capins)

The framed samples were midiprepped and will be used for transfection

Sequencing result of pSB1C3_email and pBAD_Affibody_middlelinker_EGFP_His

Sequencing showed that the PCR reaction we designed to remove the mutations in the z34c insert were successful! Both pSB1C3_587_KO_Z34C and pSB1C3_587_KO_Z34C_Spacer are correct. We hadn't sent the 587_Z34C for sequencing because the colony PCR hadn't shown any bands. But because this construct is less important for cell culuture, we won't continue working on this construct.

Cloning of Darpin into pCerulean_Vp1up_NLS and pSB1C3

Investigator: Achim

The Darpin needs to be cloned into pSB1C3 for submission to the parts registry as well as into pCerulean for expression.

Darpin ( cut with NgOMIV & SpeI

Vectors cut with AgeI & SpeI

-> Results in fusion protein

Cloning of pSB1C3_001_CMV and pSB1C3_001_Vp2/3_HSPG-Ko_CMV_ZEGFR:1907

Investigator: AnissaComment: By working with three-fragment-ligation, CMV and ZEGFR:1907 will each be cut out of pSB1C3, will be ligated together and into the vector pSB1C3_001_Vp2/3_HSPG-Ko. Additionally, CMV will be cloned freom pSB1C3 into pSB1C3_001

test digestion of all CD constructs

Investigator: Kira

Comment: Because the PCR failed also with new suffix primer, we decided to check if the DNA samples are ok. 3 different CD samples were used as DNA samples.
1_original CD with iGEM binding sites p(223)
2_CD with SDM of PstI
3_CD with SDM of PstI and NgoMIV (p290)

components

volume in µl

DNA

3

BSA 10x

2,0

Buffer 4

2,0

PstI-HF

0,5

NgoMIV

1,0

H2O

11,5

Total volume (e.g. 20 µl)

20

Cloning of pSB1C3_Affibody_middlelinker_EGFP_His

Investigator: JessicaComment: There was a mistake in cloning pSB1C3_Affibody_middlelinker_EGFP_His so there is just pSB1C3_EGFP_His. in this plasmid will be cloned Affibody_middlelinker_EGFP_His

Comments: Yesterday, 2 clones of each construct were picked. Today mini-prep and test digestion will be performed of one of each approach - the other clones will be spinned down and stored at -20°C - waiting for test digestion and sequencing results.

Comment:First test digestion using XmaI and SpeI did not work out, thererfore, two different approaches using MngBI/PstI and SnaBI and PstI were performed. This verified successful cloning of P5tataless into the target vector.

Sent for sequencing:

pSB1C3_001_RC_ICRK_P5tataless_RFC10 clone 1 (P628) was sent for sequencing using VR2 primer.

mGMK_TK30 approaches

Investigator: Bea

Comment: Since we are still waiting for the TK30 gene ordered at Mr.Gene we decided to take another road to produce a BioBrick compatible thymidine kinase gene. Therefore, we are cloning the mGMK_TK30 fusion construct into the pSB1C3 and after removing the PstI site (see next topic) it is ready for submitting it to the parts registry. Additionally, we are fusing the mGMK_TK30 to the composite part pSB1C3-leftITR_PROMOTER_betaglobin for testing it in cell culture with pur final super construct.

Digestion of the constructs:

P51.2 = pSB1C3_CFP

P81 = pAAV_RFC25_mGMK_TK30

P434 = pSB1C3_leftITR_CMV_betaglobin

P437 = pSB1C3_leftITR_phTERT_betaglobin

Components

vP51.2 /µL

vP81 upstream/µL

vP81 /µL

vP434/µL

vP437/µL

DNA

5

5

10

4,5

4,5

BSA (10x)

2

2

2

2

2

Buffer no. 4 (10x)

2

2

2

2

2

Enzyme 1

EcoRI

SpeI

SpeI 1

-

-

Enzyme 2

SpeI

XbaI

EcoRI

SpeI

SpeI

H2O

9

9

4

10,5

10,5

Total volume

20

20

20

20

20

Overview cloning plan:

Loading plan of a 0,8% agarose gel:

M P51.2 P81 (digested with E+S) P81 (digested with X+S) P434 P437

Expected sizes of the digested products are:

pAAV_RFC25_mGMK_TK30= 4626, 1717bp

pAAV_RFC25_mGMK_TK30= 4626, 1717bp

pSB1C3_CFP = 2052, 756bp

pSB1C3_leftITR_CMV_betaglobin= 5094b

pSB1C3_leftITR_phTERT_betaglobin= 5094b

Results:

After Gel extraction have been performed the ligation was conducetd:

Design of mutagenesis primers for deletion of PstI in mGMK_TK30

Investigator: Bea

Comment: Since we are still waiting for the TK30 gene ordered at Mr.Gene we decided to take another road to produce a BioBrick compatible thymidine kinase gene. Therefore, new primers were designed in order to remove the PstI restriction sites within the TK30 gene. The primers will be ordered tomorrow.

Cloning of Darpin into pCerulean_Vp1up_NLS and pSB1C3 Part Two

Investigator: Achim

I accidentally plated yesterdays trafo with the wrong antibiotics and I had to digest pSB1C3 again anyway, so I prepared a new digestion of both vectors with Age & Spe.

After watching the gel picture I realized I had digested pSB1C3 with the wrong enzymes. I should have used NgOMIV and Spe in order to cut out BLA.

pCerulean_Vp1up_NLS was ligated with yesterdays Darpin fragment and plated on Ampicillin, the pSB1C3_BLA construct will be cloned correctly tomorrow

Cloning of pGGTBT7_Affibody_middlelinker_EGFP_His

Investigator: JessicaComment: pBAD is not really working, so we test another expression-plasmid

Conclusion: Except of pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_HSPG-KO (P618), all sequencing results looked well. This means: The VP2/3-HSPG-KO motif was cloned successfully to the VP2-Fusion and VP1-Insertion approaches and are ready to be tested in cell culture via qPCR and ELISA. --> Co-transfection with pSB1C3_001_RC_iRCK_VP1or2-KO_HSPG-KO + pHelper + pSB1C3_GOI.

In addition to that, clone 2 of pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_HSPG-KO will be preped and test digested tomorrow.

135. labday 30.09.2010

Cloning of Darpin into pCerulean_Vp1up_NLS and pSB1C3 Part Two

Comment: Yesterday's sequencing results revealed, that VP2/3_HSPG-KO was successfully cloned to the VP2 fusion and VP1 insertion approaches, except of pCerulean_ZEGFR:1907_MiddleLinker_VP2/3_HSPG-KO clone 2. The chromatogram showed that fusion worked, but there was a kind of contamination in the sample - already visible in the test digestion.

DNA = 2 µL

BSA (10x) = 1 µL

Buffer 4 = 1 µl

EcoRI = 0.5 µL

PstI = 0.5 µL

H20 = 5 µL

Incubation time = 45 minutes.
0.9 % Agarose gel.

Test digestion looked well. Sample was sent for sequencing.

Cloning VP2/3_Capins and VP2/3_HSPG-KO downstream of Middle Linker

Investigator: Hanna

Comment: Besides the Affibody we also want to test and characterize the DARPin for its binding properties referring to the VP2 fusion and VP1 insertion. Therefore some preliminary cloning steps have to be performed. Today the VP2/3_insCap and VP2/3_HSPG-KO sequence will be cloned downstream of the Middle Linker for the VP2 fusion.